3-Hydroxyacetaminophen, which has been identified by gas-chromatography mass spectrometry and liquid chromatography as a liver microsomal metabolite of acetaminophen, has been quantitated by a liquid chromatographic assay and a catechol O-methyl transferase plus (3H-methyl) S-adenosyl methionine assay. Its formation is apparently unrelated to the formation of the reactive toxic metabolite of acetaminophen since ascorbic acid and glutathione, both of which block covalent binding of the reactive metabolite of acetaminophen to protein, had little affect on its formation. Moreover, in microsomal incubation mixtures containing 3H-acetaminophen plus NADPH, its formation apparently does not lead to appreciable amounts of covalent binding since neither superoxide dismutase nor catechol O-methyl transferase plus S-adenosyl methionine blocked covalent binding of the radiolabeled acetaminophen to protein.